Germline IgH band is 6.2 kb, correct integration of transgene produces 9.4 kb band, indicated by asterisk in ES #80. Flow Cytometry Splenocytes and peritoneal lavage were prepared as previously described [17]. were also observed in extrafollicular clusters in the spleens of aged AM14 sd-Tg MRL/lpr mice. Class switch and antibody secretion were observed additionally in AM14 sd-Tg BALB/c B cells activated in vivo using IgG2a anti-chromatin antibodies. Development of Genistein IgG autoantibodies is a hallmark of severe autoimmunity, and is related to pathogenesis. Using the AM14 sd-Tg, we now show that switched autoantibody-forming cells develop robustly outside germinal centers, further confirming the extrafollicular expression of AID. This model will allow more physiological studies of B cell biology in the future, including memory responses marked by class switch. and [33,34]. METHODS Creation of sd-Tg Mice The targeting construct was created from the original AM14 conventional Tg, except that a 1.2 kb region of homology to the germline DH region was added at the 5 terminus and a thymidine kinase cassette was added at the 3 terminus (Figure 1A). The presence of upstream VH and DH gene segments in site-directed BCR transgenes renders them particularly susceptible to RAG-mediated VH replacement during B cell development using an internal heptamer in the 3 coding region of the BMP2 rearranged transgene [35] or the RSS segments of other downstream J segments. To improve stability of the transgene, we mutated the JH4 heptamer from 5-CACAATA (on the anti-sense strand) to 5-TGCAATA and introduced a silent mutation to mutate the internal VH heptamer from 5-CACAATA to 5-CTCAATA. Mutagenesis of RSS heptamers was performed with the Quick-Change Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturers instructions. ES cells were transfected and blastocysts injected by the Animal Genomics Service of the Yale Cancer Center, using their established protocols. PCR screening of transfected ES cell clones was performed with primers within the germline DH region (5 ATC TAC ATA GCT AGA GAG CTA GAG G 3) and the neomycin resistance cassette (5 GCA TCG CAT TGT CTG AGT AGG TGT CA 3). Southern blotting of EcoRI digests of genomic ES cell DNA was performed as described [17] with radiolabeled JH4 probe. The 1.6 kb HindIII-EcoRI fragment from the targeting construct was labeled with 32P-dCTP (Amersham), using the Random-Primed DNA Labeling Kit (Roche) according to the manufacturers instructions. Chimeric pups derived from blastocyst injection of ES cells were first bred to Fas-intact MRL/Mp mice to confirm germline transmission of the sd-Tg. AM14 sd-Tg mice were then backcrossed for 8 generations to Fas-deficient MRL/Mpmice for analysis of seroconversion. AM14 sd-Tg mice were also backcrossed at least 10 generations to the BALB/c strain. All mice were genotyped using PCR as previously described [17]. All mouse experimentation was approved by the Yale Institutional Animal Care and Use Committee. Open in a separate window Figure 1 Construction of AM14 sd-Tg mice. A) Targeting construct, germline IgH locus, and final Genistein integrated transgene are shown. Neo = neomycin resistance cassette; AM14 = AM14 rearranged AM14 VDJ3; DQ52 = Genistein 3 germline DH gene segment; J1C3 and J4 = germline JH gene segments; E = IgM enhancer; C = IgM C region exons; TK = thymidine kinase cassette. Vertical lines indicate restriction sites: EcoRI (E), HindIII (H), SalI (S), and XhoI Genistein (X). Dashed lines indicate regions of homologous recombination with germline IgH locus. White arrows indicate site-directed mutagenesis of RSS heptamers. Black arrows indicate PCR primers to screen for correct upstream integration. Striped bar indicates J4 probe used in Southern blot. B) Southern blot of EcoRI digest of genomic DNA from 3 ES cell clones which were PCR positive for correct upstream integration. Germline IgH band is 6.2 kb, correct integration of transgene produces 9.4 kb band, indicated by asterisk in ES #80. Flow.